Vitamin B12 is an important vitamin for humans and animals. It is used to treat pernicious anaemia, and peripheral neuritis, and is used as a dietary supplement. Vitamin B12 is also an important animal feed supplement as growth enhancer.
The term vitamin B12 is used to describe compounds of the cobalt corrinoid family, in particular those of the cobalamin group. The most used compound of this group is cyanocobalamin and as such the term vitamin B12 is sometimes used to refer to cyanocobalamin. In this specification the term vitamin B12 should be attributed its broad meaning so as to include all the cobalt corrinoids of the cobalamin group, which include in particular cyanocobalamin, hydroxocobalamin, methylcobalamin and 5'desoxyadenosylcobalamin characterized by a cyano, hydroxyl, methyl or 5'-desoxyadenosyl radical respectively. The methylcobalamin and 5'desoxyadenosylcobalamin compounds are known to be unstable to light in isolated form and are easily transformed to hydroxocobalamin in aqueous solution. For this reason, almost all commercial vitamin B12 preparations consist of the stable cyanocobalamin which as such is not the chemical form in which vitamin B12 can be found in nature. In this specification the term natural vitamin B12 is defined so as to comprise all chemical forms of vitamin B12 naturally occurring in nature, cyanocobalamin thus being excluded.
Vitamin B12 is produced industrially by microbial fermentation, using almost exclusively Pseudomonas denitrificans and Propionibacterium species, then converting the natural vitamin B12 into the cyanocobalamin form by chemical processes including cyanidization followed by extraction and purification steps using organic solvents (as reviewed by Spalla et al, 1989 "Microbial production of vitamin B12", In: Biotechnology of vitamins, pigments and growth factors, E. J. Vandamme ed., Elsevier, London, N.Y., pp. 257-284). The chemical conversion step and any subsequent purification steps cause this production process to be expensive, unsafe to the operators and environmentally unfriendly.
Upon ingestion, animals and humans convert cyanocobalamin to one of the natural forms of vitamin B12 such as methylcobalamin or 5'-desoxyadenosylcobalamin which are required to function as a coenzyme for several biochemical conversions (Ellenbogen, L., in: Handbook of vitamins. Nutritional, biochemical and clinical aspects; ed; L. J. Machlin, Marcel Dekker Inc., New York and Basel). In addition efficient growth of animals requires the presence of a sufficient amount of vitamin B12 activity in the animal feed. Vitamin B12 preparations are frequently sold as feed supplements as such or as part of a premix containing additional vitamins and other feed additives.
It is clear therefore that a direct supply of 5'-desoxyadenosylcobalamin or methylcobalamin instead of cyanocobalamin would be of benefit. In view of the instability of these two compounds they are not produced in isolated form but are produced in dry formulation together with the biomass of the organism in which they are produced. Such formulations are well suited for use as animal feed supplement. For the production of methylcobalamin and 5'desoxyadenosylcobalamin, bacteria of the genus Propionibacterium are most preferred, because, unlike P.denitrificans, bacteria of this genus have obtained the GRAS (Generally Recognized As Safe) status from the U.S. Food and Drug Administration and are not known to produce endotoxins. Propionibacterium species are aerotolerant, nonmotile, nonsporulating, Gram-positive bacteria characterized further by the production of large amounts of propionic acid from carbohydrates, lactic acid and polyhydroxy alcohols. The genus Propionibacterium falls into the high "GC" subdivision of the Gram-positive bacteria (T. D. Brock, M. T. Madigan, J. M. Martinko and J. Parker in: The Biology of Microorganisms 7th Edition. Prentice-Hall International Inc., 1994).
Patent application RU 2001953 (Antibiotics Enzymes Research Technical Institute) refers to a spray-drying method for Propionibacterium shermanii which has been used to produce 5'-desoxyadenosylcobalamin-containing biomass. Methylcobalamin in biomass from methanogenic bacteria is commercially available from the Gedeon Richter company. However, the vitamin B12 content in these preparations in limited by the level at which the vitamin B12 is produced during fermentation and, as a consequence, the vitamin B12 content in these preparations does not exceed 0.1% (w/w). The concentration of vitamin B12 in a composition is customarily measured an the dry weight of vitamin B12 as a percentage of the dry weight of the composition.
Concentrated (i.e. &gt;0.1% w/w) products of cyanocobalamin are commercially available. However, feed mills and premix manufacturers cannot use this material because as a consequence of its electrostatic properties cyanocobalamin segregates from its carrier upon processing in feed mills and premix manufacturers when formulated at concentrations&gt;0.1% w/w. Segregation in a particular problem during sieving, wind sifting or allowing the composition to stand for a prolonged period time.
Given these difficulties encountered in the prior art, a need remains for improved methods for the production as well as use of compositions comprising high concentrations of vitamin B12 activity. The present invention thus seeks to provide such improved methods which avoid if not completely overcome some of the problems of the prior art. In particular, it is an object of the present invention to provide a process for isolating natural vitamin B12 in relatively concentrated form. These new concentrated products preferably can be used, for example, in animal feed, particularly human food, or as an ingredient in cosmetics. The new products may be more convenient to use and to is transport (i.e., inasmuch as they are concentrated), thus allowing a reduction in costs. They may advantageously be used by feed mills and premix manufacturers as they show reduced segregation of the vitamin B12 from the carrier during processing. Moreover, the process can be carried out on an industrial scale and is relatively environmentally friendly, as there is no need to use organic solvents or cyanidation. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.